Dihydrostreptomycin and acid addition salts



gram-negative organisms,

Patented Feb. 21, 1950 DIHYDROSTBEPTOMYCIN AND ACID ADDITION SALTSRobert L. Peck, Plainfield, N. J., assignor to Merck 8: 00., Inc.,Rahway, N. J., a corporation of New Jersey No Drawing. ApplicationFebruary 15, 1946, Serial No. 648,007

. 13 Claims. (01. 260-205) This invention relates to processes forpreparing new therapeutically useful anti-biotic substances, andparticularly to the new anti-biotic substance, dihydrostreptomycin, andacid salts thereof and the process for preparing the same by catalyticreduction of acid salts of streptomycin.

Streptomycin, which is a water-soluble, levorotatory, thermostable,nitrogenous, basic organic substance exhibiting therapeutically usefulproperties, is elaborated by strains of the micro-organism, Actinomycesgriseus, in suitable culture media (Proc. Soc. Expl. Biol. Med, 55,66-69 (1944)). Streptomycin is strongly bacteriostatic againstgram-positive organisms, including Bacillus mycoides and Bacilluscereue, and against including Pseudomonas fluorescens, Pseudomonasaeroginosa and Serratia marcescens.

Streptomycin is obtained from the elaboration product of the A. griseusby adsorption on activated carbon followed by elution with an acidicsolvent in a manner similar to that used for streptothricin (Proc. Soc.Expl. Biol. Med.,' 49, 207-212 (1942) The crude product thus obtained isa streptomycin acid salt such as streptomycin hydrochloride of lowactivity or potency. The activity or potency of this anti-biotic is ameasure of the purity of the material and is determined by biologicalmethods in terms of the inhibition of growth of micro-organisms such asE. coli and B. subtilis in comparison with a standard of known activity.The-present procedure for assaying streptomycin is the same as that usedfor streptothrycin (J. Bact., 45, 408-409 (April 1943) The crudestreptomycin acid salt can be purifled-by repeated selective adsorptionand elution using an adsorbent medium such as acid-washed aluminum oxidein accordance with a process fully disclosed in my pending applicationSerial No. 601,337, filed June 23, 1945. By this procedure alone, or byusing this procedure followed by preparation of the crystallinehelianthine salt of streptomycin and subsequent conversion thereof todesired mineral acid salts in accordance with the procedure fullydescribed in the pending application of my colleague Karl Folkers, Se-

rial No. 601,335, filed June 23, 1945, now Patent No. 2,462,175,substantially pure acid salts of streptomycin are obtained. These acidsalts contain three equivalents of acid per mole of streptomycin base.

While the streptomycin acid salts thus obtained are in a form suitablefor therapeutic use, it is found that these compounds have certaininher- 2 ent disadvantages including a degree of chemical instabilityevidenced particularly in media containing components capable ofreacting with carbonyl groups.

It is now discovered, accordingto the present invention, that when anacid salt of streptomycin is subjected to the action of hydrogen in thepresence of a noble metal catalyst, hydrogen is adsorbed in the ratio ofone mole per mole of streptomycin salt thereby forming the correspondingdihydrostreptomycin compound in substantially quantitative yield. Thedihydrostreptomycin compounds are approximately as activeanti-biotically as the streptomycin salts and are equally suitable forclinical application. The essential advantage of the dihydrostreptomycincompounds over previously known streptomycin salts resides in thegreater stability of the dihydrostreptomycin compounds in the presenceof substances capable of reacting with carbonyl groups.

' The salts of the two anti-biotic substances are scarcelydistinguishable analytically, but they are readily distinguished by thefact that dihydrostreptomycin salts are stable for 24 hours in a slightexcess of hydroxylamine in buffered aqueous solution at pH 4 whilecorresponding streptomycin acid salts are completely inactivated by thisreagent.

Regarded in certain of the broader aspects, novel features of thepresent invention comprise the new and therapeutically usefulanti-biotic substance of the class consisting of dihydrostreptomycin andacid salts thereof and the process for preparing the same by catalyticreduction of acid salts of streptomycin. The acid salts contemplated inthe present invention are acid salts in which the acid moiety eitherresists reduction or, if reduced, does not react with or preventreduction of streptomycin during the reduction process. Suitable acidsalts are mineral acid salts and organic acid salts in which the acidmoiety contains no reducible groups other than C=C bonds.

In carrying out the process in accordance with the present invention aquantity of an acid salt of streptomycin is dissolved in water in whichis suspended a noble metal catalyst such as platinum metal, platinumoxide, palladium metal, and the like and a mixture is placed under ahydrogen atmosphere and shaken or agitated for a time sufilcient toconsume approximately one mole of hydrogen per mole of streptomycin saltemployed at atmospheric pressure. In general, the reaction is fasterwith platinum than with palladium, using a given amount of catalyst. Theamount of catalyst used is not critical in the reaction although therate of the reduction is increased or decreased with an increase ordecrease in the amount of catalyst. It will also be understood that thetime for completing the reaction can be shortened by conducting thereaction at increased pressure.

As starting materials, it is possible to use impure concentrates ofstreptomycin salts, but it is preferred to use a substantially pure acidsalt such as streptomycin trihydrochloride for the process.

After completion of the reduction, the catalyst is removed by filtrationand the dihydrostreptomycin compound is isolated, preferably by freezingthe filtrate and drying under vacuum from the frozen state. The freebase, dihydrostreptomycin, can be prepared from an acid salt bysubjecting a solution of the acid salt to a reaction with a base orbasic oxide which forms substantially quantitatively an insoluble saltwith the acid ion of the streptomycin salt. Thus, for example, reactingan aqueous solution of dihydrostreptomycin sulfate with thestoichiometric equivalent of barium hydroxide precipitates the sulfateas insoluble barium sulfate which is readily removed by filtration andthe free base can then be recovered from the filtrate by precipitationfrom suitable aqueous-organic solvent mixture or by freeze-drying.

The empirical formulae for streptomycin and dihydrostreptomycin arebelieved to be Example I About 50 mgm. of plantinum oxide (PtOz-2Hz0)suspended in cc. of water is reduced with hydrogen and 48.0 mgm. ofstreptomycin hydrochloride is added. A hydrogen atmosphere is maintainedand the mixture is agitated for about one and one-half hours, duringwhich time approximately one molar equivalent of hydrogen is absorbed bythe streptomycin hydrochloride in addition to that absorbed bythecatalyst. The catalyst is then removed by filtration and the filtrate isfrozen and dried under vacuum from the frozen state. Dihydrostreptomycinhydrochloride is thus recovered as a white powder which is further driedat about 56. C. in vacuo over P205 for two hours. This product has anactivity of about 762 units per mgm. and exhibits an optical rotation a=88.7 (c, 1% water).

The corresponding helianthine salt is prepared by adding a warm'aqueoussolution of methyl orange to dihydrostreptomycin hydrochloride dissolvedin methanol. Orange crystals with metallic luster separate and theproduct obtained after two recrystallizations from aqueous methanol hasan activity of about 350 units/mgm. and the following partial analysis:Calculated for C21H41N7O12(C14H15N3Q|S)at C, 50.46; H, 5.79; N, 14.94.Found: C, 50.22; H. 5.76; N. 15.17.

Example 11 About 255 mgm. of platinum oxide, suspended in 125 cc. ofwater containing one drop of 2 N hydrochloric acid, is reduced withhydrogen, and 1.396 gm. of streptomycin hydrochloride is added. Themixture is agitated in a hydrogen atmosphere for about one hour, duringwhich time approximately one molar equivalent of hydrogen is consumedper mole of streptomycin trihydrochloride inaddition to .that absorbedby the catalyst. The catalyst is then removed by filtration and thesolution is frozen and dried from the frozen state, giving aquantitative yield of dihydrostreptomycin hydrochloride as a whitegranular solid having an activity of about 750 units/mgm. and exhibitingan optical rotation =84 (c, 1% in water). A partial analysis Of thisproduct is as follows: Calculated for C21H41N7O12'3HC12 N, 14.15; Cl.15.35. Found:' N, 13.91; Cl, 15.36.

A portion of the dihydrostreptomycin hydrochloride is converted to thecrystalline helianthine salt by the procedure given in Example I, andthe product thus obtained has the following partial analysis: Calculatedfor C21H41N7012- (C14Hl5N303s) a c, 50.59; H, 5.53. Found: 0, 50.89; 11,5.75.

Example III Equals amounts of treptomycin hydrochloride anddihydrostreptomycin hydrochloride are treated with slightly more than amolar equivalent of hydroxylamine buffered to pH 4 and allowed to standfor 24 hours at 25 C. At the end of this period, the anti-bioticactivity of the streptomycin salt is completely destroyed, while theactivity of the dihydrostreptomycin salt remains approximately 750units/mgm.

Example IV About 1.5 gm. of streptomycin sulfate is dissolved in about125 cc. of water and 0.3 gm. of

platinum catalyst (PtO2'2H2O) is added. The

mixture is shaken mechanically while a hydrogen atmosphere is maintainedin the reaction vessel.

( C21H41N'1Q12) 2 3H2SO4 Example V About 2.9 gm. of streptomycinhydrochloride is dissolved in 100 cc. of water and 0.3 gm. of palladiummetal catalyst, prepared according to Willstatter and Waldschmidt-Leitz,Ben, 54, 123 (1921), is added. The mixture is placed under a hydrogenatmosphere and shaken mechanically. Hydrogen uptake is slow and afterabout two hours shaking, 0.5 gm. of palladium catalyst is added toincrease the rate of reduction. Ap-

proximately one mole of hydrogen per mole of 0 streptomycinhydrochloride is absorbed at the end of about 24 hours shaking. Thecatalyst is filtered oil and the filtrate is frozen and dried undervacuum from the frozen state, yielding dihydrostreptomycin hyrochlorideas a white granular solid having an activity of about 700 units/mgm. andan optical rotation 02D =-82 (c, 1.0% in water).

Example VI To about 428 mgm. of dihydrostreptomycin sulfate in about 5cc. of water is slowly added 3.8 cc. of 0.465 N barium hydroxidesolution. The barium sulfate formed is removed by filtration. Tests forbarium and sulfate ions in the clear, colorless filtrate are negative.The filtrate is then mixed with one volume of acetone causing slowseparation of a micro-crystalline precipitate weighing about 110 mgm.The supernatant solution is mixed with about volumes of acetone causingthe separation of a second crop of white micro-crystalline materialweighing about 150 mgm. Both crops of the free base,dihydrostreptomycin, show an activity of about 750 units/mgm. and anoptical rotation a =92 (c, 1% in pH 7.0 buffer solution). When heated onthe micro-block, crystals of the free base begin to develop a slightyellow color at about 130 C. and melt to a soft mass at 195-205" C. withthe formation of an orange-brown color and with the evolution of a smallamount of gas. The empirical formula for the free base is C21H41N7O12.

Modifications may be made in carrying out the present invention withoutdeparting from the spirit and scope thereof, and my invention is to belimited only by the appended claims.

I claim:

1. The process that comprises reacting an acid salt of streptomycin withhydrogen in the presence of a hydrogenation catalyst and recovering thecorresponding salt of dihydrostreptomycin from the reaction mixture.

2. The process that comprises reacting an aqueous solution of an acidaddition salt of streptomycin with hydrogen in the presence of ahydrogenation catalyst until substantially one mole of hydrogen per moleof streptomycin is taken up and recovering from the reaction mixture thecorresponding acid salt of dihydrostreptomycin.

3.- The process that comprises reacting an acid salt of streptomycinwith hydrogen in the presence of platinum oxide catalyst and recoveringfrom the reaction mixture the corresponding 5 acid salt ofdihydrostreptomycin.

4. The process that comprises reacting streptomycin hydrochloride withhydrogen in the presence of a hydrogenation catalyst and recovering fromthe reaction mixture dihydrostreptomycin hydrochloride.

5. The process that comprises reacting streptomycin sulfate withhydrogen in the presence of a hydrogenation catalyst and recovering fromthe reaction mixture of dihydrostreptomycin sulfate.

6. The process that comprises reacting a substantially pure acid salt ofstreptomycin with hydrogen in the presence of a hydrogenation catalyst,removing the salt component of the dihydrostreptomycin acid salt thusformed by adding the stoichiometric equivalent of a base which forms aninsoluble compound with said salt component, filtering off the insolublecompound, and recovering from the filtrate the free base,dihydrostreptomycin.

7. The process that comprises reacting substantially pure streptomycinsulfate with hydrogen in the presence of a hydrogenation catalyst,removing the sulfate component of the dihydrostreptomycin sulfate thusformed by adding the stoichiometric equivalent of barium hydrate andprecipitating barium sulfate, filtering off the precipitate, andrecovering from the filtrate the freebase, dihydrostreptomycin.

ROBERT L. PECK.

No references cited.

8. A substance of the class consisting of dihy-

1. THE PROCESS THAT COMPRISES REACTING AN ACID SALT OF STEPTOMYCIN WITHHYDROGEN IN THE PRESENCE OF A HYDROGENATION CATALYST AND RECOVERING THECORRESPONDING SALT OF DIHYDROSTREPTOMYCIN FROM THE REACTION MIXTURE. 8.A SUBSTANCE OF THE CLASS CONSISTING OF DIHYDROSTREPTOMYCIN AND ACIDSALTS THEREOF.